MODULE Common Staining Technique Microbiology 2 Notes COMMON STAINING TECHNIQUE 2.1 INTRODUCTION Staining is technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biological tissues for viewing, often with the aid of different microscopes. Stains may be used to define and examine bulk tissues (highlighting, for example, muscle fibers or connective tissue), cell populations (classifying different blood cells, for instance), or organelles within individual cells. Bacteria have nearly the same refractive index as water, therefore, when they are observed under a microscope they are opaque or nearly invisible to the naked eye. Different types of staining methods are used to make the cells and their internal structures more visible under the light microscope. Microscopes are of little use unless the specimens for viewing are prepared properly. Microorganisms must be fixed & stained to increase visibility, accentuate specific morphological features, and preserve them for future use OBJECTIVES After reading this lesson, you will be able to: z describe the need for staining techniques z explain terms related to staining techniques z discuss the substances used as stain z enlist various staining techniques z classify & explain various stains 20 MICROBIOLOGY Common Staining Technique MODULE 2.2 TERMS RELATED TO STAINING Microbiology Stain A stain is a substance that adheres to a cell, giving the cell color. The presence of color gives the cells significant contrast so they are much more visible. Different stains have different affinities for different organisms, or different parts of organisms. They are used to differentiate different types of organisms or to view specific parts of organisms Notes Staining Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes. Fixation Fixation by itself consists of several steps–aims to preserve the shape of the cells or tissue involved as much as possible. Sometimes heat fixation is used to kill, adhere, and makes them permeable so it will accept stains What can be used as stain The substance be used as a stain must be colored or it should react in the system to give a colored product, because of which some portion of the system becomes colored and the rest remains colorless. Staining renders the organism more visible, it displays the structure and finer details of bacteria and it helps to differentiate between organisms Staining techniques Direct staining - The organism is stained and background is left unstained Negative staining - The background is stained and the organism is left unaltered INTEXT QUESTIONS 2.1 Fill in the blanks: 1. Staining is primarily used to enhance ................ in the image 2. Substances that adhere to cell giving colour to cell are called as ................ 3. ................ aims to preserve the shape of the cells 4. The organism is stained and background is left unstained in ................ staining technique 5. Background is stained and the organism is left unchanged in ................ staining technique MICROBIOLOGY 21 MODULE Common Staining Technique Microbiology 2.3 KINDS OF STAINS Stains are classified as z Simple stain z Differential stain z Structural or special stains Notes Simple Staining The staining process involves immersing the sample (before or after fixation and mounting) in dye solution, followed by rinsing and observation. Many dyes, however, require the use of a mordant, a chemical compound that reacts with the stain to form an insoluble, coloured precipitate. When excess dye solution is washed away, the mordanted stain remains. Simple staining is one step method using only one dye. Basic dyes are used in direct stain and acidic dye is used in negative stain. Simple staining techniques is used to study the morphology better, to show the nature of the cellular contents of the exudates and also to study the intracellular location of the bacteria Commonly used simple stains are z Methylene blue z Dilute carbol fuchsin z Polychrome methylene blue Loeffler’s Methylene Blue Method of Staining Flood the smear with methylene blue, allow for 2 minutes, pour off the stain and allow the air to dry by keeping in a slanting position and by this the organism will retain the methylene blue stain Use Methylene blue staining is used to make out clearly the morphology of the organisms eg. H.influenzae in CSF, Gonococci in urethral pus Polychrome Methylene Blue Preparation Allow Loeffler’s Methylene blue to ‘ripen’ slowly. Methylene blue stain is kept in half filled bottles, aerate the content by shaking at intervals, Slow oxidation of methylene blue forms a violet compound and Stain gets polychrome property. The ripening nearly takes 12 months and this is hastened by addition of 1% potassium carbonate 22 MICROBIOLOGY Common Staining Technique MODULE Use Microbiology Polychrome Methylene Blue is used to demonstrate Mc Fadyean reaction of B.anthracis and in this the blue bacilli Is surrounded by purple capsular material Dilute Carbol Fuchsin Preparation Prepare carbol fuchsin and dilute it to 1/15 using distilled water Notes Method of staining Flood the smear and let stand for 30 seconds, wash with tap water and blot gently to dry Use To stain throat swab from patients of suspected Vincent’s angina, (Borrelia are better stained), it is used as a counter stain in Gram stain and to demonstrate the morphology of Vibrio cholerae (comma shaped) INTEXT QUESTIONS 2.2 Fill in the blanks: 1. Chemicals that reacts with stain to form precipitates are called as .............. 2. .............. dyes is used in direct stain 3. .............. dyes is used in negative stain 4. Commonly used simple stains are .............., .............. & .............. 2.4 DIFFERENTIAL STAINS Gram Staining Differential Stains use two or more stains and allow the cells to be categorized into various groups or types. Both the techniques allow the observation of cell morphology, or shape, but differential staining usually provides more information about the characteristics of the cell wall (Thickness). Gram staining (or Gram’s method) is an emprical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell wall. The Gram stain is almost always the first step in the identification of a bacterial organism, While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique, thus forming Gram variable and Gram MICROBIOLOGY 23 MODULE Common Staining Technique Microbiology indeterminate groups as well. The word Gram is always spelled with a capital, referring to Hans Christian Gram, the inventor of Gram staining Gram staining Principles Gram staining is used to determine gram status to classify bacteria broadly. It is based on the composition of their cell wall. Gram staining uses crystal violet Notes to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. Gram status is important in medicine; the presence or absence of a cell wall will change the bacterium’s susceptibility to some antibiotics. Gram-positive bacteria stain dark blue or violet. Their cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria Gram Staining Technique 1. Crystal violet acts as the primary stain. Crystal violet may also be used as a simple stain because it dyes the cell wall of any bacteria. 2. Gram’s iodine acts as a mordant (Helps to fix the primary dye to the cell wall). 3. Decolorizer is used next to remove the primary stain (crystal violet) from Gram Negative bacteria (those with LPS imbedded in their cell walls). Decolorizer is composed of an organic solvent, such as, acetone or ethanol or a combination of both.) 4. Finally, a counter stain (Safranin), is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization Gram Reaction Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counter stain (safranin or fuchsine) and appearing red or pink. Gram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall. Gram- positive cell walls typically lack the outer membrane found in Gram-negative bacteria. Gram-negative bacteria are those bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counter stain (commonly safranin) is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color. The test itself is useful in classifying two distinct types of bacteria based on the structural differences of their cell walls. On the other hand, Gram-positive bacteria will retain the crystal violet dye when washed in a decolorizing solution. 24 MICROBIOLOGY Common Staining Technique MODULE Microbiology Notes Fig. 2.1: Gram Reaction. MICROBIOLOGY 25 MODULE Common Staining Technique Microbiology INTEXT QUESTIONS 2.3 Fill in the blanks: 1. Gram staining uses ............... to stain cell walls, ............... as mordant & ............... as counter stain Notes 2. Gram positive bacteria stains ............... colour 3. Gram negative bacteria stains ............... colour 4. Gram positive organism are able to retain crystal violet stain because of high amount of ............... in the cell wall 2.5 ACID-FAST STAINING The Ziehl–Neelsen stain, also known as the acid-fast stain, widely used differential staining procedure. The Ziehl – Neelsen stain was first described by two German doctors; Franz Ziehl (1859 to 1926), a bacteriologist and Friedrich Neelsen (1854 to 1894) a pathologist. In this type some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid- fast organisms.